The overall goal of this Program Project Grant is to understand the genetic and metabolic defects that produce hypercholesterolemia and accelerated atherosclerosis in man. Over the past 15 years, we have defined a new pathway for the control of cholesterol metabolism in human cells. This pathway, called the low density lipoprotein (LDL) receptor pathway, consists of an ordered sequence of events by which cells bind LDL at the surface receptor site, internalize the lipoprotein by endocytosis, degrade it in lysosomes, and release its cholesterol for use in the synthesis of cell membranes, steroid hormones, bile acids, and plasma lipoproteins. The importance of the LDL pathway is illustrated by the demonstration that mutations at discrete steps in this sequence produce human diseases involving hypercholesterolemia and atherosclerosis. We now apply for a 5-year renewal of our Program Project Grant (Years 16- 20) that will allow us to further pursue the integrated, multidisciplinary approach that is currently in operation. We propose to focus on 4 key molecules with established roles in cholesterol homeostasis: LDL receptor (which controls LDL levels in plasma), HMG CoA reductase and HMG CoA synthase (which control cholesterol synthesis in cells), and cholesterol 7alpha-hydroxylase (which controls formation of file acids in liver). We will also investigate 4 other proteins with potentially important roles in cholesterol homeostasis: LDL receptor-related protein (LRP) (which may function to remove chylomicrons from plasma), oxysterol binding protein (which may act as a regulator of intracellular sterol homeostasis), farnesyl:protein transferase (which farnesylates proteins that may participate in posttranscriptional regulation of HMG CoA reductase), and Lp(a) (which accelerates atherosclerosis). A series of model systems will be used to study the mechanisms by which these 8 proteins operate at the molecular level (i.e., the gene, the mRNA, and the protein), at the level of the intact cell, and at the level of the whole animal. We will use a multidisciplinary approach, including a wide variety of techniques, involving biochemistry, immunology, molecular biology, molecular genetics, cell biology, electron microscopy, animal physiology (including transgenic mice), and clinical genetics. The successful completion of these studies outlined in the Program Project Grant should enhance understanding of the molecular basis of hypercholesterolemia and atherosclerosis.